Positive modulator of bone morphogenic protein-2

ABSTRACT

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/064,039, filed on Feb. 22, 2005, now issued as U.S. Pat. No.7,482,427, the specification of which is incorporated herein byreference, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/547,012, filed on Feb. 20, 2004, thespecification of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support undercontract number DE-AC02-98CH10886, awarded by the U.S. Department ofEnergy. The United States Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Field of the Invention (Technical Field)

The present invention relates to synthetic growth factor modulatorcompositions, particularly modulators of the Bone Morphogenic Protein(BMP) family. Compositions of the present invention are of the formulasdisclosed herein with a single or dual chain peptide sequence havingspecific binding affinity to a BMP-2 receptor, a linker, optionally ahydrophobic linker, and a non-growth factor heparin-binding sequence,and methods of use of synthetic growth factor modulators.

Background Art

Note that the following discussion refers to a number of publications byauthor(s) and year of publication, and that due to recent publicationdates certain publications are not to be considered as prior artvis-à-vis the present invention. Discussion of such publications hereinis given for more complete background and is not to be construed as anadmission that such publications are prior art for patentabilitydetermination purposes.

Bone Morphogenic Proteins (BMPs) are a group of proteins involved in thedevelopment of a wide range of organs and tissues from embryonic throughadult stages, (Wozney J M 2002, Spine 27(16 Suppl 1):S2-8). BMPs alsoplay important roles in tissue repair and remodeling processes followinginjuries. Certain BMPs induce ectopic bone formation and enhance healingof critical-sized segmental bone defects in animal models. Clinicalstudies show that recombinant human BMPs (rhBMPs) are safe and effectivealternatives to autologous bone grafting. rhBMP-2 and rhBMP-7 areapproved for human use in spinal fusion and recalcitrant long-bonenonunions, respectively. (Kleeman et al. 2001, Spine 26(24):2751-6.Burkus et al. 2002, Spine 27(21):2396-408. McKay et al. 2002, Spine27(16 suppl 1):S66-85. Poynton et al. 2002, Spine 27(16 suppl 1):S40-8.)

The effectiveness of rhBMP-2 seems to heavily depend on the dose.Significantly higher-than-physiological doses are required fortherapeutic effect in vivo. For example, levels in the neighborhood of 1mg/mL of rhBMP-2 are used in spinal fusion cages (up to 8 mg/cage), anamount three orders of magnitude higher than what is typically foundendogenously. (McKay et al. 2002, Spine 27(16 suppl 1):S66-85.)Administration of such a high dose of recombinant protein is not onlycostly, but may also be associated with adverse effects such as bonyovergrowth and immunological reactions. Therefore, the development ofpositive modulators of BMP-2 to enhance BMP activities is of clinicalsignificance.

BMP-2 signaling involves two types of transmembrane serine/threoninekinase receptors, namely type I (BRI) and type II (BRII). (Hoodless etal. 1996, Cell 85(4):489-500. Kawabata et al. 1995, J Biol Chem270(10):5625-30. Nohno et al. 1995, J Biol Chem 270(38):22522-6.Rosenzweig et al. 1995, Proc Natl Acad Sci USA 92(17):7632-6.) An activeligand/receptor complex consists of BMP-2, BRI, and BRII in a 2:2:2ratio. (Reddi A H 2001, J Bone Joint Surg Am 83-A Suppl 1 (Pt 1):S1-6.)Both type I and type II receptors are required for BMP-2 to exert itsbiological functions. Upon BMP-2 binding, BRI kinase is activated as aresult of phosphorylation by BRII. BRII would not bind to BMP-2 withoutthe presence of BRI and the complex of BMP-2 and BRII is not capable ofinitiating signaling in the absence of BRI. The serine/threonine kinasein the BRI receptor is believed to be responsible for thephosphorylation of Smad1, Smad5, and Smad8, which in turn assemble intoheteromeric complexes with Smad4 and translocate into the nucleus toregulate transcription of target genes. (Massague et al. 2000, Genes Dev14(6):627-44. Attisano et al. 2000, Curr Opin Cell Biol 12(2):235-43.)In addition, the activated receptor complexes can activate the p38 MAPkinase pathway independent of the Smad pathway. (Iwasaki et al. 1999, JBiol Chem 274(37):26503-10. Miyazono K 2000, J Cell Sci 113(Pt7):1101-9.) Currently there are thought to be two modes for BMP-2 toinitiate signaling. Gilboa and colleagues showed that multiple BMPreceptor oligomers are present at the cell surface prior to ligandbinding. (Gilboa et al. 2000, Mol Biol Cell 11(3):1023-35.) Nohe andcolleagues then showed that the pre-formed receptor complexes areresponsible for the BMP-2 induced Smad pathway activation, andBMP-2-induced receptor complexes initiate the p38 kinase pathway. (Noheet al. 2002, J Biol Chem 277(7):5330-8.)

Some efforts have been made to generate heparin-binding growth factoranalogs. For example, natural platelet-derived growth factors (PDGF)occur as an A chain and a B chain arranged in head-to-head (AA or BB)homodimers, or (AB or BA) heterodimers. Thus, U.S. Pat. No. 6,350,731 toJehanli et al. discloses PDGF analogs in which two synthetic PDGFreceptor-binding domains are covalently linked through a polyglycine oran N-(4-carboxy-cyclohexylmethyl)-maleimide (SMCC) chain to mimic thenatural active polypeptide dimer.

U.S. Pat. No. 6,235,716 to Ben-Sasson discloses analogs of angiogenicfactors. The analogs are branched multivalent ligands that include twoor more angiogenic homology regions connected by a multilinker backbone.

U.S. Pat. No. 5,770,704 (the '704 patent) to Godowski disclosesconjugates for activating receptor tyrosine kinases, cytokine receptorsand members of the nerve growth factor receptor superfamily. Theconjugates include at least two ligands capable of binding to thecognate receptor, so that the binding of the respective ligands inducesoligomerization of these receptors. The ligands disclosed in the '704patent are linked by covalent attachment to various non-proteinaceouspolymers, particularly hydrophilic polymers, such as polyvinylalcoholand polyvinylpyrrolidone, and the polyvinylalkene ethers, includingpolyethylene glycol and polypropylene glycol. The ligands includehepatocyte growth factor (HGF) peptide variants that each bind HGFreceptor, thereby causing receptor dimerization and activation of thebiological activity of the HGF receptor dimer.

U.S. Pat. No. 6,284,503 (the '503 patent) to Caldwell et al. discloses acomposition and method for regulating the adhesion of cells andbiomolecules to hydrophobic surfaces and hydrophobic coated surfaces forcell adhesion, cell growth, cell sorting and biological assays. Thecomposition is a biomolecule conjugated to a reactive end groupactivated polymer. The end group activated polymer includes a blockcopolymer surfactant backbone and an activation or reactive group. Theblock copolymer may be any surfactant having a hydrophobic regioncapable of adsorbing onto a hydrophobic surface, and a hydrophilicregion which extends away from the surface when the hydrophobic regionis adsorbed onto the hydrophobic surface. The '503 patent discloses thatthe biomolecules that may be conjugated to the end group activatedpolymer include natural or recombinant growth factors, such as PDGF,EGF, TGFα, TGFβ, NGF, IGF-I, IGF-II, GH and GHRF, as well asmulti-CSF(II-3), GM-CSF, G-CSF, and M-CSF.

Other workers have described compositions that include homologs andanalogs of fibroblast growth factors (FGFs). See for example U.S. Pat.No. 5,679,673 to Lappi and Baird; U.S. Pat. No. 5,989,866 to Deisher etal. and U.S. Pat. No. 6,294,359 to Fiddes et al. These disclosuresrelate to FGF homologs or analogs that are either conjugated to a toxicmoiety and are targeted to the FGF receptor-bearing cells; or arehomologs or analogs that modulate the biological pathways through thesignal transduced by the FGF receptor upon binding by the FGF homolog oranalog.

A series of patent applications to Kochendoerfer et al. disclosepolymer-modified proteins, including synthetic chemokines anderythropoiesis stimulating proteins. See, for example, InternationalPublications WO 02/04105, WO 02/19963 and WO 02/20033. These includechemically ligated peptide segments of a polypeptide chain of asynthetic erythropoiesis protein, such that a polypeptide chain results,with a water soluble polymer attached at one or more glycosylation siteson the protein. These applications also disclose synthetic chemokines,which are also polymer modified, and are asserted to be antagonists.However, heparin-binding domains are not disclosed. Other erythropoietinmimetics are known, such as those disclosed in U.S. Pat. Nos. 5,773,569and 5,830,851 to Wrighton et al.

International Publication WO 00/18921 to Ballinger and Kavanaughdiscloses a composition consisting of fusion proteins having FGFreceptor affinity linked to an “oligomerization domain”, either directlyor through a linking group. The oligomerization domain ranges in lengthfrom about 20 to 300 residues, and includes constructs such astranscription factors, Fc portions of IgG, leucine zippers and the like.The oligomerization domains disclosed are homodimeric domains, wherein asingle FGF receptor affinity fusion protein is linked to a singledomain, such as a leucine zipper, which in turn is linked to a similarmolecule by means of cysteine residues at both the amino and carboxytermini of the leucine zippers, such that two parallel leucine zippers,each with a single FGF receptor affinity fusion protein, arecross-linked by means of disulfide bonds. It is also disclosed thatfusion proteins may include a heparin binding domain, such as the use ofjun as a multimerization domain, which is asserted to be a heparinbinding domain. Thus the compositions disclosed by Ballinger andKavanaugh are all composed of a single receptor-binding sequencecovalently attached to an oligomerization domain, whereby two or moresimilar oligomerization domains, each with a single receptor-bindingsequence, are conjoined by means of either an association provided bythe oligomerization domain, or alternatively, are chemicallycross-linked to provide for the covalent bonding of the individualcomponents.

The above described homologs, analogs, conjugates or ligands eachinclude a receptor-binding domain. However, none of the disclosedcompounds or compositions further include both a linker, providing forthe linking of two receptor-binding domains to a dipeptide sequence, andfurther providing a single non-signaling peptide containing aheparin-binding domain. Moreover, none of these or other knownheparin-binding growth factor analogs provide the advantages describedherein below. Further, the prior art does not disclose modulators which,through a synergistic effect, increase or enhance the efficacy of anaturally occurring growth factor, such as BMP-2.

BRIEF SUMMARY OF THE INVENTION

Compounds of the present invention are partial agonists of bonemorphogenic protein 2 (BMP-2), and particularly human BMP-2. As usedherein, “BMP-2” includes specifically human BMP-2, but is not limited tohuman BMP-2. Compounds of the present invention substantially augmentthe bioactivity of BMP-2. Among other applications, compounds of thepresent invention can be employed as an additive to demineralized bonematrix (DBM) and bone graft materials to maximize the bioactivity ofBMP-2. Compounds of the present invention augment the bioactivity ofBMP-2 found in DBM (exogenous) and in bone undergoing repair(endogenous). Compounds of the present invention are preferably made bysolid phase peptide chemistry. The clinical use of compounds of thepresent invention provide a new and novel treatment strategy applicableto accelerating bone repair, among other uses.

Compounds of the present invention substantially increase thebio-effectiveness of BMP-2 and significantly decrease the BMP-2 dosethreshold. Compounds of the present invention plus BMP-2 result insignificant increases of alkaline phosphatase (ALP) activity atsub-threshold concentrations of BMP-2. Compounds of the presentinvention interact directly with BMP receptor isoforms, and thecombination of compounds of the present invention and BMP-2 causes asynergistic repression of mitogen-activated protein kinase (MAP kinase)and a synergistic increase of Smad activation. The synergistic increaseof Smad activation is hypothesized to be largely responsible for theobserved effect or action of these compounds on a system. Compounds ofthe present invention may be supplied with DBM, for example, withenhanced bone repair accordingly resulting from a) the augmentationBMP-2 found in DBM, and b) augmentation of host BMP-2 known to beunregulated in bone-repair. Similarly, if compounds of the presentinvention are supplied in concert with classic osteoconductive materialssuch as tricalcium phosphate or calcium sulfate, it can augment hostBMP-2 and lead to osteoinduction and increased cellular migration intothe bone fill material. Both approaches take advantage of the fact thatBMP-2 and its receptors are up-regulated during bone repair processes.

One embodiment of the present invention is a compound of formula I:

wherein:

-   -   X is a peptide chain that (i) has a minimum of three amino acid        residues, (ii) has a maximum of about fifty amino acid residues,        and (iii) binds specifically to a Bone Morphogenic Protein-2        receptor;    -   R₁ is independently hydrogen, such that the terminal group is        NH₂, an acyl group with a linear or branched C₁ to C₁₇ alkyl,        aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or is amino acid, a dipeptide or a tripeptide with        an N-terminus NH₂, NH₃ ⁺, or NH group;    -   R₂ is independently a trifunctional alpha amino acid residue,        wherein X is covalently bonded through a side chain of R₂;    -   R₃ is independently a linker comprising a chain from 0 to about        15 backbone atoms covalently bonded to R₂;    -   R₄ is OH such that the terminal group is a carboxyl, NH₂, an        acyl group with a linear or branched C₁ to C₁₇ alkyl, aryl,        heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or NH—R₁;    -   Y is a linker comprising a chain from 0 to about 50 backbone        atoms covalently bonded to R₂ and Z; and    -   Z is a non-signaling peptide chain that includes a heparin        binding domain comprising an amino acid sequence that        comprises (i) a minimum of one heparin binding motif, (ii) a        maximum of about ten heparin binding motifs, and (iii) a maximum        of about thirty amino acids.

Yet another embodiment of the present invention is a bioactive implantcontaining a coating of formula I. Yet another embodiment of the presentinvention is a medicament for the therapeutic or prophylactic treatmentof treat bone lesions or degenerative joint conditions made from formulaI. Still another embodiment of the present invention is a compound offormula I used in a pharmaceutical composition and or a pharmaceuticallyacceptable salt thereof and a pharmaceutical carrier.

Another embodiment of the present invention is a compound of formula II

wherein:

-   -   X is a peptide chain that (i) has a minimum of three amino acid        residues, (ii) has a maximum of about fifty amino acid residues,        and (iii) binds specifically to a Bone Morphogenic Protein-2        receptor;    -   R₁ is independently hydrogen, such that the terminal group is        NH₂, an acyl group with a linear or branched C₁ to C₁₇ alkyl,        aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or is amino acid, a dipeptide or a tripeptide with        an N-terminus NH₂, NH₃ ⁺, or NH group;    -   R₆ is independently a linker comprising a chain from 0 to about        15 backbone atoms covalently bonded to R₅ when the linker is        greater than 0 atoms;    -   R₅ is a trifunctional alpha amino acid residue, wherein X is        covalently bonded through a side chain of R₅;    -   R₄ is OH such that the terminal group is a carboxyl, NH₂, an        acyl group with a linear or branched C₁ to C₁₇ alkyl, aryl,        heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or NH—R₁;    -   Y is a linker comprising a chain from 0 to about 50 backbone        atoms covalently bonded to R₅ and Z; and    -   Z is a non-signaling peptide chain that includes a heparin        binding domain comprising an amino acid sequence that        comprises (i) a minimum of one heparin binding motif, (ii) a        maximum of about ten heparin binding motifs, and (iii) a maximum        of about thirty amino acids.

Another embodiment of the present invention is a bioactive implanthaving at least one coating containing the compound of formula II.

Yet another embodiment of the present invention is a pharmaceuticalcomposition containing the compound of formula II or a pharmaceuticallyacceptable salt thereof and a pharmaceutical carrier.

Yet another embodiment of the present invention is a method to enhancebone formation or to treat bone lesions or to treat degenerative jointconditions in a vertebrate animal, which method comprises administeringto a vertebrate subject in need of such treatment an effective amount ofa compound of formula I or formula II that augments Bone MorphogenicProtein-2 activity wherein the compound is a synthetic peptide having anon-growth factor heparin binding region, a linker and a sequence thatbinds specifically a to Bone Morphogenic Protein-2 Receptor.

One aspect of the present invention provides a synthetic growth factormodulator.

Another aspect of the present invention provides a compound that is asynthetic growth factor analog which is a positive modulator of BMP-2activity in vivo.

Yet another aspect of the present invention provides a compound that isa positive modulator of BMP-2 activity in vitro.

Still another aspect of the present invention provides a compound thatreduces the effective dose of exogenously applied BMP-2 for therapeuticpurposes.

Another aspect of the present invention is to reduce the therapeuticallyeffective dose of recombinant BMP delivered to a subject in needthereof.

Another aspect of the present invention provides a method for treating asubject having a bone injury, by providing a compound of the presentinvention in combination with a recombinant member of the BMP family toa fracture site.

Another aspect of the present invention provides a method for treating asubject having a bone injury, by providing a compound of the presentinvention to a fracture site.

Another aspect of the present invention provides a method for treating asubject in need of bone growth, by providing a compound of the presentinvention in combination with a recombinant member of the BMP family toa site in a subject in need of treatment.

Another aspect of the present invention provides a method for treating asubject in need of bone growth, by providing compound of the presentinvention to a site in a subject in need of treatment.

Another aspect of the present invention provides for kits containing acompound of the present invention.

Another aspect of the present invention provides for kits containing acomposition of the present invention.

Another aspect of the present invention is a bioactive implantabledevice containing a compound of the present invention.

Other aspects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention.

The objects and advantages of the invention may be realized and attainedby means of the instrumentalities and combinations particularly pointedout in the appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIGS. 1A and 1B are graphs illustrating that B2A2 enhances BMP-2induction of alkaline phosphatase (ALP) activity in C3H10T½ cells.

FIG. 2 is a graph illustrating that B2A2 enhances the activity ofrecombinant human BMP-2 obtained from CHO cell and E. coli commercialproduction methods.

FIG. 3 is a graph illustrating that the synergistic effect of B2A2 wasspecific to BMP-2.

FIG. 4 is a graph illustrating the induction of ALP activity despite thetemporal separation of the addition of B2A2 and BMP-2 to the C2C12 cellline.

FIG. 5 is a graph illustrating that B2A2-coated surfaces enhanced BMP-2activity. Surfaces of a variety of compositions were first coated withsilyl heparin under sterile conditions in tissue culture dishes (a 1%solution in acid ethanol incubated 30 min at 37° C., rinsed with H₂O,dried at 56° C.).

FIG. 6 is a graph illustrating the relative density from radiographicimage analysis from athymic rats implanted at 3 weeks.

FIG. 7 is a graph illustrating the relative number of L6 cells inculture after treatment with cytotoxic agents or B2A2-K—NS.

FIG. 8 is a graph illustrating the induction of osteogenicdifferentiation in C2C12 cells with varying concentrations of B2A2-K—NSin the presence and absence of BMP-2.

FIG. 9 is a graph comparing the area of explants excised from an areaimplanted with matrigel containing B2A2-K—NK analog with and withoutBMP-2.

FIG. 10 is a graph illustrating Alcian staining of chondrogenic pathwayproteins in C3H10T½ cells whose expression was stimulated by B2A2-K—NStreatment.

FIG. 11 is a graph illustrating the induction of osteogenicdifferentiation in C2C12 cells by B2A7-K—NS in the presence and absenceof suboptimal concentration of BMP-2.

FIG. 12 is a graph illustrating the specific binding between a compoundof the present invention and BMP-2 Receptor.

FIG. 13 is a graph illustrating the synergistic action of BMP-2 and B2A2binding to BMP-2 receptor.

DETAILED DESCRIPTION OF THE INVENTION

In a clinical setting, compounds of the present invention may besupplied with DBM, for example, with enhanced bone repair accordinglyresulting from a) the augmentation BMP-2 found in DBM, and b)augmentation of host BMP-2 known to be upregulated in bone-repair.Similarly, if compounds of the present invention are supplied in concertwith classic osteoconductive materials such as tricalcium phosphate, itcan augment host BMP-2 and lead to osteoinduction and increased cellularmigration into the bone fill material. Both approaches take advantage ofthe fact that BMP-2 and its receptors are up-regulated during bonerepair processes.

In keeping with the known activation pathway of BMP-2, it ishypothesized that compounds of the present invention interact directlywith BMP receptor isoforms (BRI and BRII), and that the combination of acompound of the present invention and BMP-2 causes a synergisticrepression of mitogen-activated protein kinase (MAP kinase) and asynergistic increase of Smad activation compared to using BMP-2 alone.While BMP-2 inhibitors are known, these are the first known BMP-2enhancers that functions in the physiological range.

Compounds of the present invention interact directly with BMP receptorsto positively modulate BMP-2 induced events leading to osteogenicdifferentiation. Synergistic effects between compounds of the presentinvention and BMP-2 were observed in two multipotent cell lines, C3H10T½and C2C12, as determined by at least two osteogenic differentiationmarkers, ALP activity and phosphorylation of Smad. The augmentation ofALP activity at any given concentration of BMP-2 was generally a 5-20fold increase. While researchers have identified other BMP-2 modulatorsthat have either been negative regulators or agents that fail to workunder normal physiological conditions, compounds of the presentinvention are the first peptide specific regulators that positivelymodulate BMP-2.

Recently several BMP-specific antagonists have been identified. Noggin,chordin, and gremlin have been shown to bind to BMPs with the sameaffinity as BMP receptors, and thus competitively inhibit BMPs.(Zimmerman et al. 1996, Cell 86(4):599-606. Hsu et al. 1998, Mol Cell1(5):673-83.) In a rat marrow cell culture, bFGF has been shown to actsynergistically with BMP (Hanada et al. 1997, J Bone Miner Res12(10):1606-14. Wang et al. 1993, Acta Orthop Scand 64(5):557-61.),however, higher doses of bFGF caused profound inhibitory effect in vivo.Spinella-Jaegle and colleagues reported that Sonic hedgehog (Shh)enhanced BMP-2 effects in C3H10T½ and ST2 cells, but it failed toenhance BMP-2 activity in analogous osteoprogenitor cells C2C12 and apreosteoblast cells MC3T3-E1. They further showed that the enhancingeffect appeared to be a priming effect in which Shh increased thepercentage of cells responding to BMP-2 (Spinella-Jaegle S, et al. 2001,J Cell Sci 114(Pt 11):2085-94), whereas Shh itself is able to induce ALPactivity in C3H10T½. (Nakamura et al. 1997, Biochem Biophys Res Commun237(2):465-9. Kinto et al. 1997 FEBS Lett 404(2-3):319-23. Katsuura etal. 1999, FEBS Lett 447(2-3):325-8. Yuasa et al. 2002, J Cell Physiol193(2):225-32.)

In another line of investigation, attempts to generate peptides thatpossess BMP activity have been less than satisfactory. Osteoinductiveeffects were reported by Dee and colleagues for a stretch of BMP-7sequence (White et al. 2001, vol. BED-Vol. 50. American Society ofMechanical Engineers, Snowbird, Utah, pp 201-202.), and also by Suzuki &Tanihara for two overlapping stretches of BMP-2 sequence (Saito et al.2003, Biochim Biophys Acta 1651(1-2):60-7. Suzuki et al. 2000, J BiomedMater Res 50(3):405-9.). These results, however, were obtained insupranormal experimental systems with peptides at extremely highconcentrations and/or covalently attached to a substrate that kept themin contact with cells for a period of weeks. For example, the linearpeptide reported to have the highest BMP-2-like activity (Saito et al.2003, Biochim Biophys Acta 1651(1-2):60-7.) works only at concentrations˜2,000 times higher than BMP-2—at this level it completely displacesBMP-2 from cell surface receptors and is thus a competitor of BMP-2.

In contrast to prior-art peptides, compounds of the present inventionenhance the activity of BMP-2 and do so in a concentration range ofBMP-2 that can be anticipated in physiological settings.

Different sources of BMPs present different attributes to consider forhuman applications. BMPs have been purified from bone, but with very lowyields, and potential health risks associated with isolation fromallogenic donor bone also limit clinical application of BMP from thissource. Most of the BMP in clinical use is recombinant protein obtainedfrom eukaryotic cell culture. Complications of post-translationmodification and low yield result in a very high cost of theserecombinant proteins. Moreover, the amounts required for efficacy inhuman applications turned out to be unexpectedly high (McKay et al.2002, Spine 27(16 suppl 1):S66-85. Poynton et al. 2002, Spine 27(16suppl 1):S40-8.).

A BMP-specific enhancer, such as that disclosed herein, has uniqueclinical significance. A BMP-2 enhancer may be used to reduce theamounts of BMP-2 required. This is of medical and practical significancebecause as a synthetic peptide, compounds of the present invention are(a) less expensive to produce, (b) vastly more chemically stable, and(c) easy to chemically modify for enhanced drug delivery. Biologically,there are also other advantages. For example, the process of spinalfusion involves a sequence of events associated with a temporal andspatial pattern of osteogenic-related gene expression. Morone andcolleagues (Morone et al. 1998, Clin Orthop (351):252-65.) studied theexpression of the mRNA of several BMPs in spinal fusion and found thatBMP-2 and others were increased at different levels at different times.It is daunting to match exogenous application recombinant BMP-2 to thebiologically optimal schedule. Similarly, BMPs can occur as homo- andheterodimers. A BMP enhancer may thus be effective by augmenting thenatural endogenous expression of BMPs as they occur in situ.

Compounds of the present invention can thus be used to reduce theeffective dose of recombinant BMP-2 on or associated with medicaldevices, to maximize the biological activity of biological preparationslike demineralized bone matrix (DMB), and to augment the endogenouslevels of BMP-2 generated by host tissue during bone healing process.

DBM is one alternative material that is bone-derived and widely used inclinical practice. DBM is processed from human bone via solvent and acidtreatments, and in its final form contains collagens and low levels ofgrowth factors. DBM is available from a number of companies andorganizations, including Wright Medical Technologies, Osteotech, theAmerican Red Cross, and Innova. DBM, via the collagen component,provides a scaffold on which new bone forms and it also has someosteoinductive potential via its low levels of growth factors. It mayalso elicit some activation of mesenchymal stem cells from thesurrounding area that differentiate into osteoblasts.

The osteoinductive potential of DBM is low, however, and varies widelyfrom lot-to-lot and manufacturer-to-manufacturer. Since the growthfactors in DBM are expected to have their most pronounced effect onosteoprogenitor cells, the availability of osteoprogenitor cells iscritical when demineralized bone matrix is used. The limited ability ofDBM to elicit a robust osteoinduction is widely seen as a limitingfactor in the use of this material.

Among the calcium-rich bone graft materials, there are a large number ofcommercially available products bone filler agents that are not derivedfrom human sources, including Pro Osteon™ (coralline hydroxyappatite,Interpore Cross International), Bioglass™ (bioactive glass implant, USBiomaterials Corp.), Collagraft™ (hydroxyapatite/tricalcium phosphateand pure bovine fibrillar collagen, Zimmer), Cellplex™ (tricalciumphosphate, synthetic cancellous bone, Wright Medical Technologies,Inc.), and a number of calcium phosphate and calcium phosphate fillers.All of these materials are osteoconductive and support the in-growth ofcapillaries, perivascular tissues, and osteoprogenitor cells from a hostinto an implant or graft. They are not, however, osteoinductive.

Among the biologics, a number of companies have developed bone fillproducts that are intended to be used with autologous bone marrow cellsor platelet concentrates. These products are intended to increase thenumber of stem cells in a graft or to increase the amount of growthfactors, respectively.

In a related vein, the InFuse™ spinal cage product (Sofamor-Danek, adivision of Medtronic) is an example of a device that combines aosteoconductive material (collagen) with an osteoinductive agent.InFuse™ is indicated for use in conjunction with spinal fusionprocedures, and a similar product is being developed for fresh fracturerepair.

The success of InFuse™, and to a lesser extent, Stryker Corporation'sOP-1™ for use in tibial non-unions, has led to a high level of interestin recombinant growth factor approaches. Numerous additional growthfactors are being evaluated in the orthopedic and ortho-biologic fields.Yet among the various BMPs, BMP-2 appears to be the factor with thehighest degree of osteoinduction.

There is thus an increasing clinical demand for bone graft materials anda high level of interest in alternatives to growth factors orimprovements in existing bone graft materials.

DEFINITIONS

As used here and elsewhere, the following terms have the meanings given.

The term “alkene” includes unsaturated hydrocarbons that contain one ormore double carbon-carbon bonds. Examples of such alkene groups includeethylene, propene, and the like.

The term “alkenyl” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one double bond; examplesthereof include ethenyl, 2-propenyl, and the like.

The “alkyl” groups specified herein include those alkyl radicals of thedesignated length in either a straight or branched configuration.Examples of such alkyl radicals include methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl,isohexyl, and the like.

The term “aryl” includes a monovalent or bicyclic aromatic hydrocarbonradical of 6 to 12 ring atoms, and optionally substituted independentlywith one or more substituents selected from alkyl, haloalkyl,cycloalkyl, alkoxy, alkylhio, halo, nitro, acyl, cyano, amino,monosubstituted amino, disubstituted amino, hydroxy, carboxy, oralkoxy-carbonyl. Examples of an aryl group include phenyl, biphenyl,naphthyl, 1-naphthyl, and 2-naphthyl, derivatives thereof, and the like.

The term “aralkyl” includes a radical—R^(a)R^(b) where R^(a) is analkylene (a bivalent alkyl) group and R^(b) is an aryl group as definedabove. Examples of aralkyl groups include benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like. The term “aliphatic”includes compounds with hydrocarbon chains, such as for example alkanes,alkenes, alkynes, and derivatives thereof.

The term “acyl” includes a group RCO—, where R is an organic group. Anexample is the acetyl group CH₃CO—.

A peptide or aliphatic moiety is “acylated” when an alkyl or substitutedalkyl group as defined above is bonded through one or more carbonyl{—(C═O)—} groups. A peptide is most usually acylated at the N-terminus.

An “amide” includes compounds that have a trivalent nitrogen attached toa carbonyl group (—CO.NH₂).

An “amine” includes compounds that contain an amino group (—NH₂).

A “diamine amino acid” is an amino acid or residue containing tworeactive amine groups and a reactive carboxyl group. Representativeexamples include 2,3 diamino propionyl amino acid, 2,4 diamino butylicamino acid, lysine or ornithine.

A “trifunctional amino acid” is an amino acid or residue with threereactive groups, one the N-terminus amine, a second the C-terminuscarboxyl, and the third comprising all or a part of the side chain.Trifunctional amino acids thus include, by way of example only, diamineamino acids; amino acids with a reactive sulfhydryl group in the sidechain, such as mercapto amino acids including cysteine, penicillamine,or 3-mercapto phenylalanine; amino acids with a reactive carboxyl groupin the side chain, such as aspartic acid and glutamic acid; and aminoacids with a reactive guanadium group in the side chain, such asarginine.

Compounds of the Present Invention

According to one embodiment of the present invention, compounds are offormula I:

wherein:

-   -   X is a peptide chain that (i) has a minimum of three amino acid        residues, (ii) has a maximum of about fifty amino acid residues,        and (iii) binds specifically to Bone Morphogenic Protein-2        receptor;    -   R₁ is independently a hydrogen, such that the terminal group is        NH₂, an acyl group with a linear or branched C₁ to C₁₇ alkyl,        aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or is amino acid, a dipeptide or a tripeptide with        an N-terminus NH₂, NH₃ ⁺, or NH group;    -   R₂ is independently a trifunctional amino acid residue, wherein        X is covalently bonded through a side chain of R₂;    -   R₃ is independently a linker comprising a chain from 0 to about        15 backbone atoms covalently bonded to R₂;    -   R₄ is OH such that the terminal group is a carboxyl, NH₂, an        acyl group with a linear or branched C₁ to C₁₇ alkyl, aryl,        heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or NH—R₁;    -   Y is a linker comprising a chain from 0 to about 50 backbone        atoms covalently bonded to R₂ and Z; and    -   Z is a non-signaling peptide chain that includes a heparin        binding domain comprising an amino acid sequence that        comprises (i) a minimum of one heparin binding motif, (ii) a        maximum of about ten heparin binding motifs, and (iii) a maximum        of about thirty amino acids.        According to another embodiment of the present invention        compounds are of formula II:

wherein:

-   -   X is a peptide chain that (i) has a minimum of three amino acid        residues, (ii) has a maximum of about fifty amino acid residues,        and (iii) binds specifically to Bone Morphogenic Protein-2        receptor;    -   R₁ is independently a hydrogen, such that the terminal group is        NH₂, an acyl group with a linear or branched C₁ to C₁₇ alkyl,        aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or is amino acid, a dipeptide or a tripeptide with        an N-terminus NH₂, NH₃ ⁺, or NH group;    -   R₆ is independently a linker comprising a chain from 0 to about        15 backbone atoms covalently bonded to R₅;    -   R₅ is a trifunctional amino acid residue, wherein a first X is        covalently bonded through a side chain of R₅ and a second X is        covalently bonded through the N-terminus amine;    -   R₄ is OH such that the terminal group is a carboxyl, NH₂, an        acyl group with a linear or branched C₁ to C₁₇ alkyl, aryl,        heteroaryl, alkene, alkenyl or aralkyl chain including an        N-terminus NH₂, NH₃ ⁺, or NH group or a corresponding acylated        derivative, or NH—R₁;    -   Y is a linker comprising a chain from 0 to about 50 atoms        covalently bonded to R₂ and Z; and    -   Z is a non-signaling peptide chain that includes a heparin        binding domain comprising an amino acid sequence that        comprises (i) a minimum of one heparin binding motif, (ii) a        maximum of about ten heparin binding motifs, and (iii) a maximum        of about thirty amino acids.

In each of formula I and formula II, the covalent bonds can be, forexample, a peptide bond or other amide bond, a thioether bond or esterbond. A group is covalently bonded to another group when it is, directlyor through one or more other groups or atoms comprising covalent bonds,covalently bonded.

The chain of atoms of the Y region of formula I is covalently attachedto R₂ and to sequence Z, and in formula II the Y region is covalentlyattached to R₅ and to sequence Z. The covalent bonds can be, forexample, peptide, amide, thioether or ester bonds. Particularlypreferred is a peptide bond. Preferably, the Y region includes a chainof a minimum of about nine backbone atoms. More preferably, the Y regionincludes a chain of a minimum of about twelve backbone atoms. Mostpreferably, the Y region includes a chain of a minimum of about fifteenbackbone atoms. For example, the Y region may be formed from a chain ofat least four, at least five or at least six amino acids. Alternatively,the Y region may be formed from a chain of at least one, at least two,or at least three amino carboxylic acids, such as aminohexanoic acidresidues. Particularly preferred are embodiments in which Y is one ormore straight chain amino carboxylic acids, such as where Y comprises[NH₂—(CH₂)_(p)CO]_(q) wherein p is from 1 to about 10 and q is from 1 toabout 20. Examples of straight chain amino carboxylic acids that may beemployed include 6-aminohexanoic acid, 7-aminoheptanoic acid,9-aminononanoic acid and the like.

Preferably, the Y region includes a chain of a maximum of about fiftyatoms. More preferably, the Y region includes a chain of a maximum ofabout forty-five atoms. Most preferably, the Y region includes a chainof a maximum of about thirty-five atoms. For example, the Y region maybe formed from a chain of up to about twelve, up to about fifteen, or upto about seventeen amino acids.

The amino acid sequence of the Y region is preferably an artificialsequence, i.e. it does not include any amino acid sequence of four ormore amino acid residues found in a natural ligand of a BMP receptor.

In a particular embodiment, the Y region includes a hydrophobic aminoacid residue, or a chain of hydrophobic amino acid residues. The Yregion can, for example, include one or more amino carboxylic acidresidues, such as one, two, three or more aminohexanoic acid residues.In another alternative embodiment, the Y region can include acombination of amino acid hydrophobic residues.

In another particular embodiment, the Y region of the molecule caninclude a branched or unbranched, saturated or unsaturated alkyl chainof between one and about twenty carbon atoms. In a further embodiment,the Y region can include a chain of hydrophilic residues, such as forinstance, ethylene glycol residues. For instance, the Y region caninclude at least about three, or at least about four, or at least aboutfive ethylene glycol residues.

The Z region of the molecule of formula I and formula II is aheparin-binding region and can include one or more heparin-bindingmotifs, BBxB or BBBxxB as described by Verrecchio et al. J. Biol. Chem.275:7701 (2000). Alternatively, the Z region can include both BBxB andBBBxxB motifs (where B represents lysine, arginine, or histidine, and xrepresents a naturally occurring, or a non-naturally occurring aminoacid). For example, the heparin-binding motifs may be represented by thesequence [KR][KR][KR]X(2)[KR] (SEQ ID NO:1), designating the first threeamino acids as each independently selected from lysine or arginine,followed by any two amino acids and a sixth amino acid which is lysineor arginine.

The number of heparin-binding motifs is variable. For instance, the Zregion may include at least one, at least two, at least three or up toat least five heparin-binding motifs. Where there are more than oneheparin-binding motifs, the motifs may be the same or different.Alternatively, the Z region includes up to a maximum of about tenheparin-binding motifs. In another alternative embodiment, the Z regionincludes at least four, at least six or at least eight amino acidresidues. Further, in certain embodiments the Z region includes up toabout twenty, up to about twenty-five, or up to about thirty amino acidresidues. It is to be realized that, in part, the avidity of the Zregion for heparin is determined by the particular heparin-bindingmotifs selected and the number of such motifs in Z. Thus for particularapplications both the selection and number of such motifs may be variedto provide optimal heparin binding of the Z region.

In a preferred embodiment, the amino acid sequence of the Z region isRKRKLERIAR (SEQ ID NO:2). In another embodiment, the amino acid sequenceof the Z region is RKRKLGRIAR (SEQ ID NO:3). In yet another embodiment,the amino acid sequence of the Z region is RKRKLWRARA (SEQ ID NO:4). Inyet another embodiment, the amino acid sequence of the Z region isRKRLDRIAR (SEQ ID NO:5), providing a heparin-binding motif derived froma modification of the sequence at residues 270-279 of the Jun/AP-1 DNAbinding domain (Busch et al. Trans-Repressor Activity of NuclearGlycosaminoglycans on Fos and Jun/AP-1 Oncoprotein-mediatedTranscription. J. Cell Biol. 116:31-42, 1992). In yet anotherembodiment, the amino acid sequence of the Z region is RKRKLERIARC (SEQID NO:6). The presence of a terminal cysteine residue optionally affordsthe opportunity to link other molecules, including detection reagentssuch as fluorochromes, radioisotopes and other detectable markers, tothe Z region, as well as the opportunity to link toxins, immunogens andthe like.

The synthetic bone morphogenic protein analogs of the present invention,including those of formulas I and II, include embodiments wherein the Xregion is all or a portion, or a homolog of all or a portion, of any ofthe following amino acid sequences:

AISMLYLDENEKVVL (SEQ ID NO:7)

ISMLYLDENEKVVLKNY (SEQ ID NO:8),

LYFDESSNVILKK (SEQ ID NO:9),

LYVDFSDVGWNDW (SEQ ID NO:10),

EKVVLKNYQDMVVEG (SEQ ID NO:11),

CAISMLYLDENEKVVL (SEQ ID NO:12),

AFYCHGECPFPLADHL (SEQ ID NO:13),

PFPLADHLNSTNHAIVQTLVNSV (SEQ ID NO:14), or

In a preferred embodiment the X region is the amino acid sequenceISMLYLDENEKVVLKNY (SEQ ID NO:8). More preferably the X region is theamino acid sequence LYFDESSNVILKK (SEQ ID NO:9). More preferably still,the X region is the amino acid sequence AISMLYLDENEKVVL (SEQ ID NO:7).

The inventors have surprisingly and advantageously found that in thecompounds of the present invention, including those of formulas I andII, the X region may be synthesized in a reverse direction, such thatconsidering the sequence AISMLYLDENEKVVL (SEQ ID NO:7) illustrated inthe conventional N→C orientation, and using formula I, the first aminoacid bound to the R₂ side chains is the N-terminus amino acid residue,the second amino acid bound to the N-terminus amino acid residue is the2 position residue, and so on, and the compounds nonetheless retainbiological activity and specifically bind to a BMP receptor. It may beseen that such a construct has, based on a conventional N→C orientation,a reverse sequence, in that it is the carboxyl group of the conventionalN-terminus amino acid residue that forms a peptide bond with the epsilonamine where R₂ is a diamine amino acid. Thus again employing aconventional N→C orientation, the foregoing sequences may be employed ina reverse orientation, and the resulting compound of present inventionis biologically active and may be employed as described herein.According to a preferred embodiment, the X region is the sequenceLVVKENEDLYLMSIA (SEQ ID NO:15) (again considering the sequence in theconventional N→C orientation), as disclosed in Example 2 herein. Asdescribed in Example 2, the C-terminus alanine (A) is bound to theepsilon amine of a lysine (K) in the R₂ position of formula I, theisoleucine (I) is bound by a peptide bond to the alanine, and so on.Thus the following sequence is provided, and is biologically active, asdescribed herein:

Other reverse sequences that may be employed, in whole or in part,including homologs thereto, in addition to LVVKENEDLYLMSIA (SEQ IDNO:15), include but are not limited to YNKLVVKENEDLYLMSI (SEQ ID NO:16),KKLIVNSSEDFYL (SEQ ID NO:17), WDNWGVDSFDVYL (SEQ ID NO:18),GEVVMDQYNKLVVKE (SEQ ID NO:19), LHDALPFPCEGHCYFA (SEQ ID NO:20),VSNVLTQVIAHNTSNLHDALPFP (SEQ ID NO:21), and LVVKENEDLYLMSIAC (SEQ IDNO:22).

Alternatively, in another particular aspect the invention providessynthetic BMP, TGF or GDF (growth differentiation factor) peptideanalogs with sequences as shown in Table 1 wherein the transforminggrowth factor family member peptides are particularly useful inaugmenting the activity of endogenous or artificial BMP peptides or TGFpeptides, wherein is shown (under the heading “preferred X receptorbinding domain”) the sequence forming all or part of the X region ofconstructs of any of I or II. It is to be understood that some or only aportion of any sequence listed under the heading “preferred X receptorbinding domain” may be employed, and thus the X region employed may be asubset of any sequence listed below. It is further to be understood thatthe X sequence need not be identical to all or a portion of a sequencelisted below, and may be homologous with all or a portion, such as asequence that is 80% to 95% homologous.-*

TABLE 1 CYTOKINE PREFERRED X RECEPTOR BINDING DOMAIN TGF-β1IVYYVGRKPKVEQLSNMIVRS (SEQ ID NO: 23) TGF-β2 TILYYIGKTPKIEQLSNMIVKS(SEQ ID NO: 24) TGF-β3 LTILYYVGRTPKVEQLSNMVV (SEQ ID NO: 25) BMP-2AISMLYLDENEKVVLKNYQDMVV (SEQ ID NO: 26) BMP-3 SSLSILFFDENKNVVLKVYPNMTV(SEQ ID NO: 27) BMP-β3 NSLGVLFLDENRNVVLKVYPNMSV (SEQ ID NO: 28) BMP-4AISMLYLDEYDKVVLKNYQEMVV (SEQ ID NO: 29) BMP-5 AISVLYFDDSSNVILKKYRNMVV(SEQ ID NO: 30) BMP-6 AISVLYFDDNSNVILKKYRNMVV (SEQ ID NO: 31) BMP-7AISVLYFDDSSNVILKKYRNMVV (SEQ ID NO: 32) BMP-8 ATSVLYYDSSNNVILRKARNMVV(SEQ ID NO: 33) BMP-9 ISVLYKDDMGVPTLKYHYEGMSV (SEQ ID NO: 34) BMP-10ISILYLDKGVVTYKFKYEGMAV (SEQ ID NO: 35) BMP-11 INMLYFNDKQQIIYGKIPGMVV(SEQ ID NO: 36) BMP-12 ISILYIDAANNVVYKQYEDMVV (SEQ ID NO: 37) BMP-13ISILYIDAGNNVVYKQYEDMVV (SEQ ID NO: 38) BMP-14 ISILFIDSANNVVYKQYEDMVV(SEQ ID NO: 39) BMP-15 ISVLMIEANGSILYKEYEGMIA (SEQ ID NO: 40) GDF-1ISVLFFDNSDNVVLRQYEDMVV (SEQ ID NO: 41) GDF-3 ISMLYQDNNDNVILRHYEDMVV(SEQ ID NO: 42) GDF-8 INMYLFNGKEQIIYGKIPAMVV (SEQ ID NO: 43) GDF-9LSVLTIEPDGSIAYKEYEDMIA (SEQ ID NO: 44)

The term “homologous”, as used herein refers to peptides that differ inamino acid sequence at one or more amino acid positions when thesequences are aligned. For example, the amino acid sequences of twohomologous peptides can differ only by one amino acid residue within thealigned amino acid sequences of five to ten amino acids. Alternatively,two homologous peptides of ten to fifteen amino acids can differ by nomore than two amino acid residues when aligned. In another alternative,two homologous peptides of fifteen to twenty or more amino acids candiffer by up to three amino acid residues when aligned. For longerpeptides, homologous peptides can differ by up to approximately 5%, 10%,or 20% of the amino acid residues when the amino acid sequences of thetwo peptide homologs are aligned.

Particularly useful amino acid sequences as X regions of formulas I orII include homologs of fragments of naturally occurring sequences thatdiffer from the amino acid sequences of natural growth factor in onlyone or two or a very few positions. Such sequences preferably includeconservative changes, where the original amino acid is replaced with anamino acid of a similar character according to well known principles;for example, the replacement of a non-polar amino acid such as alaninewith valine, leucine, isoleucine or proline; or the substitution of oneacidic or basic amino acid with another amino acid of the same acidic orbasic character.

The R₃ regions of formula I or the R₆ regions of formula II can includea chain of atoms or a combination of atoms that form a chain. Typically,the chains are chains primarily of carbon atoms, that may alsooptionally include oxygen or nitrogen atoms, such as for example chainsof atoms formed from amino acids (e.g. amino acids found in proteins, aslisted above; naturally occurring amino acids not found in proteins,such as ornithine and citrulline; or non-natural amino acids, such asaminohexanoic acid; or a combination of any of the foregoing aminoacids). It is also contemplated that agents such as polyethylene glycol(PEG), polyethylene oxide (PEO), amino polyethylene glycol,bis-amine-PEG, and other variants of polyethylene glycol known to thoseskilled in the art can similarly be used. Particularly preferred for theR₃ or R₆ regions are chains which include an amino terminal and acarboxyl terminal, such that the chains may be utilized in standardpeptide synthesis methodologies. Examples include any amino acids, aminocarboxylic acids, preferably straight chain amino carboxylic acids, andbifunctional amino-PEG-acid spacers. Among amino acids, glycine ispreferred.

In certain embodiments of the invention, each of the R₃ regions offormula I or each of the R₆ regions of formula II can be different,although in most embodiments it is preferred that the regions beidentical. However, it is contemplated that such regions may differ; forexample, in formula II the R₅ may be a diamine amino acid, such aslysine. It is possible to utilize an orthogonal protecting group duringsynthesis to protect either the alpha amine or epsilon amine, tothereafter add one or amino acid residues or other groups to form an R₆group, and then to remove the orthogonal protecting group, and proceedwith parallel synthesis of the X groups from the deprotected amine on R₅and the terminal amine on R₆. Similar methods may be employed withformula I.

Methods of Synthesizing the Compounds of the Present Invention

The synthesis of the compounds of the present invention can be achievedby any of a variety of chemical methods well known in the art. Suchmethods include bench scale solid phase synthesis and automated peptidesynthesis in any one of the many commercially available peptidesynthesizers. Preferably, the synthesizer has a per cycle couplingefficiency of greater than 99 percent.

The compounds of the present invention can be produced by stepwisesynthesis or by synthesis of a series of fragments that can be coupledby similar well known techniques. See, for instance, Nyfeler, Peptidesynthesis via fragment condensation. Methods Mol. Biol. 35:303-16(1994); and Merrifield, Concept and early development of solid-phasepeptide synthesis. Methods in Enzymol. 289:3-13 (1997). These methodsare routinely used for the preparation of individual peptides. It ispossible to assemble the analogs of the present invention in componentparts, such as peptides constituting the X, Y and Z components thereof,and to thereafter couple such component parts to assemble the analog.See, for instance, Dawson and Kent, Synthesis of native proteins bychemical ligation. Annu. Rev. Biochem. 69:923-960 (2000); and Eom etal., Tandem ligation of multipartite peptides with cell-permeableactivity. J. Am. Chem. Soc. 125:73-82 (2003). However, in a preferredembodiment the compounds of the present invention are synthesized bysolid phase synthesis, with the C-terminus residue of the Z region offormulas I or II bound to resin, and the synthesis proceeding stepwise.Conventional protecting groups are employed as required, withdeprotection either prior to, during or following cleavage of thepeptide from the resin. By way of example only, for compounds of thepresent invention containing one or more lysine residues in addition toany at the R₂ position of formula I or the R₅ position of formula II,such additional lysine residues are conventionally protected with aprotecting group, and deprotected following synthesis.

Methods of Use of the Compounds of the Present Invention

The compounds of the present invention provide a cost effective sourceof biologically active molecules that are useful in a number of ways,including as soluble prophylactic or therapeutic pharmaceutical agents.

The compounds of the present invention are also useful as biologicallyactive agents as components of medical devices and for coating ofmedical devices, such as for instance, sutures, implants and medicalinstruments to promote biological responses, for instance, to stimulategrowth and proliferation of cells, or healing of wounds.

In one aspect, the invention provides a method and compositions fortreating a mammal with bone injury, by providing a compounds of thepresent invention, such as an analog of BMP-2. For example, suchcompounds of the present invention may be administered as apharmaceutical agent, or may be employed as an additive to bone matrixor bone graft materials.

The term “medical device” as used herein means a device that has one ormore surfaces in contact with an organ, tissue, blood or other bodilyfluid in an organism, preferably a mammal, particularly, a human.Medical devices include, for example, extracorporeal devices for use insurgery such as blood oxygenators, blood pumps, blood sensors, tubingused to carry blood, and the like which contact blood that is returnedto the patient. The term can also include endoprostheses implanted inblood contact in a human or animal body, such as vascular grafts,stents, pacemaker leads, heart valves, aneurism coils, and the like thatare implanted in blood vessels or in the heart. The term can furtherinclude devices for temporary intravascular use such as catheters, guidewires, and the like that are placed in blood vessels or the heart forpurposes of monitoring or repair. The term can further include nerveelectrodes, muscle electrodes, implantable pulse generators, implantabledrug pumps, and defibrillators. Moreover, the term medical device caninclude sutures, graft materials, wound coverings, nerve guides, bonewax, embolization particles, microbeads, dental implants, boneprostheses, bone graft materials, spinal fusion cages, bone fillers,orthopedic devices, tissue scaffolds, artificial joints or controlledrelease drug delivery devices.

The surface of the medical device can be formed from any of the commonlyused materials suitable for use in medical devices, such as forinstance, stainless steel, titanium, platinum, tungsten, ceramics,polyurethane, polytetrafluoroethylene, extended polytetrafluoroethylene,polycarbonate, polyester, polypropylene, polyethylene, polystyrene,polyvinyl chloride, polyamide, polyacrylate, polyurethane, polyvinylalcohol, polycaprolactone, polylactide, polyglycolide, polysiloxanes(such as 2,4,6,8-tetramethylcyclotetrasiloxane), natural rubbers, orartificial rubbers, or block polymers or copolymers thereof.

Methods for coating biological molecules onto the surfaces of medicaldevices are known. See for instance U.S. Pat. No. 5,866,113 to Hendrikset al., the specification of which is hereby incorporated by reference.Tsang et al. in U.S. Pat. No. 5,955,588 teach a non-thrombogenic coatingcomposition and methods for using the same on medical devices, and isincorporated herein by reference. Zamora et al. in U.S. Pat. No.6,342,591 teach an amphipathic coating for medical devices formodulating cellular adhesion composition, and is incorporated herein byreference.

The compounds of the present invention can be used for as an activeingredient in pharmaceutical compositions for both medical applicationsand animal husbandry or veterinary applications. Typically, the compoundof the present invention or pharmaceutical composition is used inhumans, but may also be used in other mammals. The term “patient” isintended to denote a mammalian individual, and is so used throughout thespecification and in the claims. The primary applications of thisinvention involve human patients, but this invention may be applied tolaboratory, farm, zoo, wildlife, pet, sport or other animals.

The compounds of the present invention may be in the form of anypharmaceutically acceptable salt. The term “pharmaceutically acceptablesalts” refers to salts prepared from pharmaceutically acceptablenon-toxic bases or acids including inorganic or organic bases andinorganic or organic acids. Salts derived from inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic salts, manganous, potassium, sodium, zinc, and thelike. Particularly preferred are the ammonium, calcium, lithium,magnesium, potassium, and sodium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like.

When the compounds of the present invention are basic, acid additionsalts may be prepared from pharmaceutically acceptable non-toxic acids,including inorganic and organic acids. Such acids include acetic,benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric,ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonicacid, trifluoroacetic acid, and the like. Acid addition salts of thecompounds of the present invention are prepared in a suitable solventfor the compound and an excess of an acid, such as hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, citric,tartaric, maleic, succinic or methanesulfonic acid. The acetate saltform is especially useful. Where the compounds of the present inventioninclude an acidic moiety, suitable pharmaceutically acceptable salts mayinclude alkali metal salts, such as sodium or potassium salts, oralkaline earth metal salts, such as calcium or magnesium salts.

The invention provides a pharmaceutical composition that includes acompounds of the present invention and a pharmaceutically acceptablecarrier. The carrier may be a liquid formulation, and in one embodimenta buffered, isotonic, aqueous solution. Pharmaceutically acceptablecarriers also include excipients, such as diluents, carriers and thelike, and additives, such as stabilizing agents, preservatives,solubilizing agents, buffers and the like, as hereafter described.

Thus the compounds of the present invention may be formulated orcompounded into pharmaceutical compositions that include at least onecompounds of the present invention together with one or morepharmaceutically acceptable carriers, including excipients, such asdiluents, carriers and the like, and additives, such as stabilizingagents, preservatives, solubilizing agents, buffers and the like, as maybe desired. Formulation excipients may include polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, PEG, PEO, mannitol, sodium chlorideor sodium citrate, as well as any number of simple sugars, includingsucrose, dextrose, lactose and the like, and combinations of theforegoing. For injection or other liquid administration formulations,water containing at least one or more buffering constituents ispreferred, and stabilizing agents, preservatives and solubilizing agentsmay also be employed. For solid administration formulations, any of avariety of thickening, filler, bulking and carrier additives may beemployed, such as starches, sugars, fatty acids and the like. Fortopical administration formulations, any of a variety of creams,ointments, gels, lotions and the like may be employed. For mostpharmaceutical formulations, non-active ingredients will constitute thegreater part, by weight or volume, of the preparation. Forpharmaceutical formulations, it is also contemplated that any of avariety of measured-release, slow-release or time-release formulationsand additives may be employed, so that the dosage may be formulated soas to effect delivery of a compounds of the present invention over aperiod of time.

In practical use, the compounds of the present invention can be combinedas the active ingredient in an admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, for example, oral, parenteral(including intravenous), urethral, vaginal, nasal, buccal, sublingual,or the like. In preparing the compositions for oral dosage form, any ofthe usual pharmaceutical media may be employed, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents and the like in the case of oral liquid preparations,such as, for example, suspensions, elixirs and solutions; or carrierssuch as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations such as, for example,powders, hard and soft capsules and tablets. The pharmaceutical formssuitable for injectable use include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that it may be administeredby syringe. The form must be stable under the conditions of manufactureand storage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, a polyol,for example glycerol, propylene glycol or liquid polyethylene glycol,suitable mixtures thereof, and vegetable oils.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1

Materials. C2C12 cells and C3H10T½ cells were purchased from AmericanType Culture Collection (Manassas, Va.). E. coli or Chinese hamsterovary (CHO) cell-derived recombinant human BMP-2 were purchased from R&DSystems (Minneapolis, Minn.). Soluble BMP-2 receptors as the recombinantBRI-Fc chimeric proteins were also obtained from R&D Systems.Endostatin-Fc, FGF-2, and VRGF were supplied by through the BiologicalResources Branch of Developmental Therapeutics Program, National CancerInstitute. TGF-beta1 was purchased from Sigma Aldrich Chemical Company.Bovine serum albumin (BSA), anti-phosphorylated MAP kinase antibody, andanti-human Fc antibody conjugated to horseradish peroxidase were fromSigma (St. Louis, Mo.). Fetal bovine serum (FBS), calf bovine serum(CBS), DMEM/F12 medium, and penicillin/streptomycin were purchased fromInvitrogen (Carlsbad, Calif.). Silyl-heparin isbenzyl-tetra(dimethylsilylmethyl)oxycarbamoyl-heparin and wassynthesized as detailed elsewhere (Zamora et al. 2002, Bioconjug Chem13(5):920-6.). In brief, silyl-heparin is made by reacting thehydrophobic groupbenzyl-tetra(dimethylsilylmethyl)-oxycarbamoyl-succinimide with heparinthereby resulting in an amphipathic heparin derivative that can beadsorbed onto hydrophobic surfaces. For coating purposes, silyl-heparinwas used as a 1% solution in 70% acidified, aqueous ethanol.

Alkaline phosphatase (ALP) Activity Assay. C2C12 cells and C3H10T½ cellswere cultured at 37° C. in a humidified atmosphere of 5% CO₂ and 95%air, with DMEM/F12 medium supplemented with 10% serum,penicillin/streptomycin. For the BMP-2 induced ALP assay, cells wereplated in 96-well (1×10⁴/well) dishes in regular growth medium.Twenty-four hours later, when the cells formed a confluent monolayer,medium was replaced with DMEM/F12, supplemented with 2% serum andcontaining indicated concentration of BMP-2 and/or B2A2. At 4-5 dayspost induction, ALP activity was determined as described by Akiyama andcolleagues (Akiyama et al. 1997, Exp Cell Res 235(2):362-9.) withmodifications. Briefly, cells were washed once with phosphate-bufferedsaline (PBS) and lysed with 0.1% Triton X 100 in 10 mM Tris HCl, pH 9.0.Protein concentration was determined using the BCA Protein Assay Kit(Pierce Biotechnology, Rockford, Ill.). Then ALP activity was measuredby adding ALP buffer (1 M diethanolamine, 0.5 mM MgCl₂, 1 mg/mLp-nitrophenylphosphate, pH 9.0), incubating in 37° C., and absorbance(405 nm) read at 15, 30 and 60 minutes using a microplatespectrophotometer (Molecular Devices, Sunnyvale, Calif.). The activitywas expressed as O.D. per mg protein per hour.

Peptide synthesis and preparation. The peptides B2A2 and B2A2-K—NS weresynthesized by conventional solid phase synthesis and purified byreverse phase HPLC on C-18, as described in Example 2 and 9.

Fractions of HPLC purified peptide were pooled, lyophilized, and storedfrozen. Aliquots of the lyophilized bulk material were used to determinethe peptide content, which was determined using a commercially availablekit (BCA, Pierce Endogen, Inc.). For most further purposes, the peptidewas dissolved in 5.5% glucose containing 0.05% Pluronic 127 to a finalconcentration of 0.5 mg/mL or 1 mg/mL, sterilize filtered through a 0.22micron filter, and lyophilized in aliquots containing 50 or 100 μg.

Receptor binding assays. Binding to BMP receptors in a solid phasebinding assay. B2A2 was absorded onto ELISA plates to saturation,soluble BMP receptor-immunoglobin Fc fusion proteins were added, andbound receptor was detected by HRP-conjugated anti-Fc antibody andcolorimetric assay, values shown are background substracted. Specificbinding of B2A2 to different receptor isoforms of the BMPR and ActivinReceptor family were tested, employing the receptor-Fc chimeras shown.Negative controls establishing specificity included unrelatedpolypeptide (e.g. insulin) adsorbed to the plates, and incubation ofunrelated chimeric protein (Endostatin-Fc), neither of which resulted inbinding of B2A2. Apparent two stage binding to BMPR-Ib was revealed byreceptor displacement experiments. Bound receptor was displaced by theaddition of rhBMP-2 at the levels indicated.

Cell growth. L6 rat skeletal myoblasts and cells from a human fetalosteoblast cell line (hFOB) (5) were used as target. Aliquots of cells(1-5×10³ cells) were seeded into wells of 96 well plates and allowed toattach for 6-24 hours. The medium was replaced with serum low (2%)medium containing peptide. Paclitaxel (100 ng/mL) and sodium azide(0.01%), if used, were included as reference materials known to inducecytotoxicity. Cultures were incubated typically for 3 days after whichtime the relative cell number was assessed using the tetrazolium saltMTS.

Cell migration. For studies involving migration across a wound margin,the cells were grown in vitro and used when approximately 90% confluent.A simulated wound was made by scraping cells away from the culturewaresurface. The cultures were rinsed to remove unbound cells, and thenincubated in DMEM:F12 medium containing 2% newborn calf serum with orwithout peptide. FGF-2 (50 ng/mL) was used as a positive controlreference material. The cells were allowed to migrate for 6 hours afterwhich the cells were fixed in buffered formalin. Migration was monitoredvia phase contrast microscopy. Migrating cells were those that hadmigrated across the site of the simulated wound margin.

In vivo Matrigel plug assay. The in vivo model involved subcutaneousimplant in young adult Fisher 344 of growth factor-reduced Matrigel withand without BMP-2 and B2A2. Animals were anesthetized before allprocedures by intra peritoneal injections of ketamine (50 mg/kg) andxylazine (5 mg/kg). Growth factor reduced Matrigel at 4° C. (liquidstate) was mixed with saline (control), BMP-2 (R&D Systems, Minneapolis,Minn.), B2A2-K—NS, or B2A2-K—NS plus BMP-2. Aliquots of 0.5 mL ofMatrigel with additives as above were injected subcutaneously on theupper flanks of the rats. The injection sites were clipped withstainless steel clips to prevent leakage. The Matrigel was kept on iceuntil the time of injection, as were the needle and syringe (to preventgelling in the needle). The animals were subsequently euthanized after14 days, the gel surgically removed, measured with calipers, and fixedin buffered formalin. Most of the explants had a generally ellipticalshape and the surface area of the ellipse was determined using theequation:Area=πabwhere a and b are ½ the width and height of the ellipse.

The fixed specimens were processed for histological examination andstained with either hemotoxylin and eosin or toluidine blue O(Histoserv, Inc., Germantown, Md.).

EXAMPLE 2

A compound of the present invention was synthesized by solid phasepeptide chemistry with the general structure of formula I wherein X is aBMP-2 receptor binding amino acid sequence having the sequenceAISMLYLDENEKVVL (SEQ ID NO:7) wherein SEQ ID NO:7 was stepwisesynthesized in parallel from R₂ trifunctional amino acids of formula Iwherein each R₂ is lysine. R₃ is 0 backbone atoms. The resultingsynthetic growth modulator analog is of the following specificstructure:

and is sometimes referred to as B2A2. In the foregoing structure, “Ahx”is 6-amino hexanoic acid, sometimes also called “6-Ahx” or “Hex”. Thesingle letters are standard amino acid single letter abbreviations forthe naturally coded amino acids. The two chains of SEQ ID NO:7 link tolysine of the R₂ position via a peptide bond with the epsilon amines ofthe lysine side chains.

EXAMPLE 3

The compound of Example 2 (B2A2) was tested in cell osteogenicdifferentiation studies to determine the analog's ability to stimulateosteogenic activity. B2A2 binds to BMP receptors, and that receptoractivation is associated with the expression of the osteogenictranscription factor Smad and repression of MAPK followed by aphenotypic transformation in which ALP is induced. Referring now to FIG.1A, the induction of osteogenic differentiation in C3H10T½ cells byBMP-2 in the presence and absence of B2A2 is illustrated. Treatment ofC3H10T½ cells with B2A2 alone (up to 10 μg/mL) only slightly increasesalkaline phosphatase (ALP) activity. However, B2A2 plus BMP-2 atsuboptimal concentrations (100 ng/mL) results in significant increasesof ALP activity. The EC₅₀ for BMP-2 is typically 300 ng/mL.

C3H10T½ cells were seeded onto 96-well plates, treated with BMP-2 aloneor in combination with B2A2 at different concentrations (solid circlesrepresent BMP-2 at 100 ng/mL, solid squares represent BMP-2 at 50 ng/mLand unshaded squares represent samples with no BMP-2). The cells wereincubated for 5 days, and then assayed for ALP activity. ALP activitywas assayed by conversion of para-nitrophenol phosphate (PNPP).

B2A2 alone had little if any effect on ALP activity in the dose rangebetween about 0.075-10.0 μg/mL as illustrated in FIG. 1A. The inductionof ALP activity was enhanced when cells are treated with 100 ng/mL ofBMP-2 together with B2A2. Co-treatment was not additive, but wassynergistic. Thus B2A2 is a partial agonist of BMP-2.

Referring now to FIG. 1B, the synergistic effect of B2A2 and BMP-2 isillustrated under conditions where the B2A2 concentration is constant atabout 1000 ng/mL while the concentration of BMP-2 is varied. Using afixed concentration of B2A2 (1 μg/mL), augmentation of ALP activity wasseen from as low as 25 ng BMP-2/mL to as high as 1000 ng BMP-2/mL. Thethreshold for BMP-2 induction of ALP starts at ˜30 ng/mL but in thepresence of 1000 ng/mL B2A2 the threshold was lowered to about 3 ng/mLBMP-2.

EXAMPLE 4

B2A2 was tested to determine whether B2A2 enhanced the biologicaleffects of CHO-produced rhBMP-2. Referring now to FIG. 2, induction ofALP activity in C2C12 cells by recombinant BMP-2 protein (rh-BMP-2) andB2A2 is illustrated. Rh-BMP-2 is commercially available from either E.coli or mammalian CHO cell production methods with slightly differentpotencies, yet B2A2 augments both types of rhBMP-2. Mouse C2C12 cellswere seeded onto 96 well plates, treated with B2A2 in combination withhuman BMP-2 derived from different sources (●/∘ CHO versus ▪/□ E. coli),incubated for 4 days, and then assayed for ALP activity as described.B2A2 was applied at 1000 ng/mL, and BMP-2 at the concentrationsindicated in the graph. B2A2 increased the efficacy of E. coli-derivedBMP-2 to levels similar to that of CHO cell-derived BMP-2, and theefficacy of CHO-derived BMP-2 is further increased by B2A2. Pointsrepresent means of quintuplicate determinations±SD.

EXAMPLE 5

B2A2 was tested in combination with other growth factors includingFGF-2, VEGF, and TGF-β1 for induction of ALP in C2C12 cells. Referringnow to FIG. 3, induction of ALP activity by BMP-2 but not various othergrowth factors in the presence of B2A2 is illustrated. Treatments ofFGF-2, TGF-B, VEGF alone failed to induce ALP in C2C12 cells in thepresence of B2A2 demonstrating that BMP-2 is the effector in thecombination of B2A2 and BMP-2. Mouse C2C12 cells were cultured asdescribed for FIG. 1A, treated with a combination of various growthfactors plus or minus B2A2, incubated for 3 days, and then assayed forALP activity as described for FIG. 1A. FGF-2 was used at 50 ng/mL, VEGFat 25 ng/mL, TGF-β1 at 50 pg/mL, BMP-2 at 50 ng/mL, and B2A2 at 1000ng/mL. Bars represent means of quintuplicate determinations±SD.

EXAMPLE 6

B2A2 was tested to determine whether temporal dissociation of B2A2+BMP-2administered to cells affected the BMP-2 induction of osteogenicactivity by the cell. Referring now to FIG. 4, the induction of ALPactivity is illustrated despite the temporal separation of the additionof B2A2 and BMP-2 to the C2C12 cell line. Co-administration of theagents is not required since serial addition of B2A2 followed by washoutand addition of BMP-2 in intervals up to one hour was effective ininducing ALP activity. Mouse C2C12 cells were cultured as before andB2A2 (1000 ng/mL) was added to some wells. After a 45 minute incubationall wells were rinsed with fresh medium and the medium was replaced. Toone set of wells, BMP-2 (200 ng/mL) was added, another set was incubatedan additional 30 min and then BMP-2 added, and finally yet another setwas incubated an additional 60 minutes and then BMP-2 added. After 5days ALP activity was measured. The synergistic effect was stillobserved despite the temporal separation of B2A2 and BMP-2administration and the washout in between. Data is the means oftriplicates±SD.

EXAMPLE 7

B2A2 was tested to determine whether spatial dissociation of B2A2 plusBMP-2 administered to cells affected the BMP-2 induction of osteogenicactivity in the cells. Referring now to FIG. 5, the induction of ALPactivity is illustrated despite the spatial separation of the additionof B2A2 and BMP-2. In FIG. 5A, a polystyrene surface of 96-well plateswere first coated by silyl-heparin (open bars), followed by a 1 μg/mLsolution of B2A2 (solid bars) in PBS for (1 hr at 37° C.) and rinsed inPBS and dried at room temperature. C2C12 cells were subsequently seededat densities that resulted in confluent monolayers, and after allowancefor attachment (1-2 hrs), BMP-2 at 50 ng/mL was added to the cultures.ALP activity was measured five days later. Data is the means oftriplicates±SD. While silyl-heparin alone potentiates BMP-2 activity,the ALP activity induced by B2A2 and BMP-2 together is more profound.

In FIG. 5B, stainless steel wafers were first coated with silyl-heparin(open bars) followed by 100 μg/mL B2A2 in PBS as a second coating (solidbars) and rinsed in PBS and dried at room temperature. Wafers werecoated separately in wells of a 24-well plate and transferred to a freshuntreated plate prior to cell seeding.

C2C12 cells were subsequently seeded at densities that resulted inconfluent monolayers, and after allowance for attachment (1-2 hrs),BMP-2 at the concentrations indicated in the graph were added to thecultures. ALP activity was measured five days later. Data is the meansof triplicates±SD. The results indicate that the enhancement of BMP-2 byB2A2 on stainless steel was profound. Similar results were observed forsilyl-heparin+B2A2 coating on titanium wafers in the presence of BMP-2.

EXAMPLE 8

B2A2 was tested to determine if B2A2 could augment demineralized bonematrix material (DBM) in an ectopic model of bone formation. Referringnow to FIG. 6, the synergistic activity of B2A2 with DBM for boneformation is illustrated. B2A2 was coated onto DBM. B2A2 (100 ng/mg or300 mg/mL) in a small volume of water (pH 4) was added to DBM (100μL/g), mixed, and air-dried at 37° C. The resultant DBM was then furtherdried overnight in a vacuum oven.

The B2A2-coated DBM was implanted into the muscle of athymic rats andthe radiographic density of the implant area is examined after 3 weeks.There was a 250% increase in relative bone density after 3 weeks incomparison to DBM without B2A2 and a 650% increase in bone density after6 weeks in comparison to DBM without B2A2 (data not shown), As indicatedin FIG. 6, there was a statistically significant increase inradiographic density in B2A2 coated-DBM muscle at both time points.

B2A2 can be employed as an additive to demineralized bone matrix (DBM)and bone graft materials to maximize the bioactivity of BMP-2. B2A2augments the bioactivity of BMP-2 found in DBM (exogenous) and in boneundergoing repair (endogenous). The clinical use of B2A2 provides a newand novel treatment strategy applicable to accelerating bone repair.

Table 2 below summarizes the biochemical interactions of B2A2, and themodulation of alkaline phosphatase, wherein modulation was monitoredusing C2C12 cells.

TABLE 2 Biochemical interactions of B2A2 Interaction with heparin YesMAP kinase phosphorylation Yes Positive modulation of alkalinephosphatase BMP-2 (E. coli) Yes BMP-2 (Chinese hamster ovary cells) YesBMP-7 (mammalian cell) No Modulation via a coating of alkalinephosphatase B2A2 coating, BMP-2 in solution Yes BMP-2 coating, B2A2 insolution Yes Silyl-heparin/BMP-2 coating, B2A2 in solution Yes

EXAMPLE 9

A compound of the present invention was synthesized by solid phasepeptide chemistry with the general structure of formula II wherein X isa BMP-2 receptor binding amino acid sequence having the sequenceAISMLYLDENEKVVL (SEQ ID NO:7) wherein SEQ ID NO:7 was stepwisesynthesized in parallel from the R₅ trifunctional amino acid of formulaII when R₆ is 0 backbone atoms and R₅ is lysine. The resulting syntheticgrowth modulator analog is of the following specific structure:

and is sometimes called B2A2-K-NS. In the foregoing structure, “Ahx” is6-amino hexanoic acid, sometimes also called “6-Ahx” or “Hex”. Thesingle letters are standard amino acid single letter abbreviations forthe naturally coded amino acids. The chain of SEQ ID NO:7 is grown fromthe alpha and epsilon amine groups of the lysine in the R₅ position. Thetheoretical molecular weight of B2A2-K-NS is 5344.

EXAMPLE 10

The synthetic growth factor analog of Example 9 (B2A2-K—NS) was testedfor deleterious effect on L6 cells. Referring now to FIG. 7, therelative number of L6 cells in culture after treatment with cytotoxicagents or B2A2-K—NS is illustrated. L6 cells were treated with 100 ng/mLof Paclitaxel or 0.01% sodium azide and the effects of these cytotoxicagents were compared to L6 cells treated with varying concentrations ofB2A2-K—NS after three days of treatment. B2A2-K—NS induced cellproliferation above control values at concentrations between 2-10 μg/mL.Similar results were observed in human fetal osteoblasts, C3H10T½ cellsand MC-3T3-E1 cells.

EXAMPLE 11

B2A2-K—NS was tested in cell osteogenic differentiation studies todetermine the ability of the synthetic growth analog to stimulateosteogenic activity. Referring now to FIG. 8, the induction ofosteogenic differentiation in C2C12 cells with varying concentrations ofB2A2-K—NS in the presence and absence of BMP-2 is illustrated. Treatmentof C2C12 cells with B2A2-K—NS alone (up to 10 μg/mL) only slightlyincreases alkaline phosphatase (ALP) activity, however, B2A2-K—NS plusBMP-2 results in significant increases of ALP activity even at normallysub-threshold concentrations of BMP-2. C2C12 cells were seeded onto96-well plates, treated with varying concentrations of B2A2-K—NS in thepresence (solid bars) and absence (open bars) of BMP-2 at 100 ng/mL. Thecells were incubated for 4 days, and then assayed for ALP activity. ALPactivity was assayed by conversion of para-nitrophenol phosphate (PNPP).B2A2-K—NS had no effect on the induction of ALP activity atconcentrations up to about 10 μg/mL. B2A2-K—NS substantially augmentsALP activity induced by suboptimal amounts of BMP-2 (100 ng/mL). Similarresults were obtaining with C3H10T½ cells.

EXAMPLE 12

B2A2-K—NS was tested for its ability to induce cells of preosteoblastorigin to migrate to a stimulated wound margin. Murine C3H10T½, MC3T3cells or hFOB were grown to near confluency in vitro. A stimulated woundwas made by scraping the cells away from the substrate. The cells wereallowed to migrate for 6 hours after which migration was monitored viamicroscopy. Statistical significance was determined using ANOVA followedby post hoc testing using multiple comparison versus control group(Dunnett's Method). FGF-2 was used as a positive control referencematerial and induced a significant increase in migrating cells comparedto controls (data not shown). Table 3 summarizes the increase inmigrating cells at the simulated wound margin induced by about 0.2 to2.0 μg/mL B2A2-K—NS.

TABLE 3 μg B2A2-K-NS/mL Mean Std Dev % of control Migrating C3H10T1/2cells/field 0.0 104.7 18.1 100 0.2 148.4 21.5 142 0.5 177.3 24.3 169 1.0214.6 34.9 205 2.0 197.4 12.5 188 Migrating MC-3T3 cells/field 0.0 162.843.3 100 0.2 251.2 37.2 154 0.5 286.7 24.0 176 1.0 297.7 34.3 183 2.0254.3 41.4 156 Migrating hFOB cells/field 0.0 92.4 33.5 100 0.2 149.725.3 162 0.5 164.7 28.1 178 1.0 192.4 33.2 208 2.0 165.9 27.6 179

EXAMPLE 13

B2A2-K—NS analog was tested for its effect in vivo. Referring now toFIG. 9, a comparison of the area of explants excised from an areaimplanted with Matrigel containing B2A2-K—NK analog with and withoutBMP-2 is illustrated. Adult rats were implanted with Matrigel with andwithout BMP-2 and B2A2-K—NS and after 14 days the residual gel wassurgically removed and measured. Nearly all of the implant sites thatreceived B2A2-K—NS, BMP-2, and BMP-2 and B2A2-K—NS had palpable sitesupon inspection whereas the control implant with carrier only had beenlargely adsorbed. Further, the explants from sites that had receivedB2A2-K—NS, BMP-2 or a combination of B2A2-K—NS plus BMP-2 hadsignificantly larger explants. The morphology of the explants differedwith differing explant compositions. Animals receiving only carrier hadresidual plugs that were small and tended to have morphology with poorcellular organization. Animals receiving B2A2-K—NS had plugs withmorphologies consistent with fibrocartilage. Animals receiving BMP-2treatments developed plugs containing increased numbers of cellsaccompanied by a moderate amount of organization that was consistentwith developing membranous ossification. In animals receiving B2A2-K—NSplus BMP-2, an increase in cell density was observed along with anorganization consistent with developing membranous ossification. Thecell density was greater than observed for controls or B2A2-K—NS butless than the cell density observed for explants from animals receivingBMP-2 alone (data not shown).

EXAMPLE 14

The B2A2-K—NS analog was tested in cell osteogenic differentiationstudies to determine the synthetic growth analogs ability to stimulateosteogenic activity. Referring now to FIG. 10, the induction ofosteogenic differentiation in C2C12 cells by B2A2-K—NS in the presenceand absence of suboptimal concentrations (100 ng/mL) of BMP-2 isillustrated. Treatment of C2C12 cells with B2A2-K—NS alone (up to 10μg/mL) only slightly increases alkaline phosphatase (ALP) activity.However, B2A2-K—NS plus BMP-2 results in significant increases of ALPactivity even at normally sub-threshold concentrations of BMP-2. C2C12cells were seeded onto 96-well plates, treated with B2A2-K—NS atdifferent concentrations in the presence (solid bars) or absence(unshaded bars) of 100 ng/mL BMP-2. The cells were incubated for 4 days,and then assayed for ALP activity. ALP activity was assayed byconversion of para-nitrophenol phosphate (PNPP).

EXAMPLE 15

The B2A2-K—NS analog was tested for its ability to induce phenotypicexpression changes in cells independent of BMP-2 (data not shown). MC3T3cells were stimulated with B2A2-K—NS and changes in the expression ofosteocalcin, osteoponin, and type II collagen were observed as measuredwith specific antibodies to each which were subsequently detected withsecondary antibodies conjugated to HPRO. The developed membranes weredigitized with a scanner and converted to gray scale with colorinversion with software.

Referring now to FIG. 10, Alcian staining of C3H10T½ cells forchondrogenic pathway derived proteins is illustrated. B2A2-K—NSincreases the amount of Alcian blue stainable material produced inC3H10T½ cells at 10 days after stimulation. Suboptimal amounts of BMP-2(50 ng/mL) did not augment the increase in Alcian blue stainablematerial.

EXAMPLE 16

A compound of the present invention was synthesized by solid phasepeptide chemistry with the general structure of formula II wherein X isa BMP receptor binding amino acid sequence having the sequenceLYFDESSNVILKK (SEQ ID NO:9) wherein SEQ ID NO:9 was stepwise synthesizedin parallel from the R₅ trifunctional amino acid of formula II when R₆is 0 atoms and R₅ is a lysine. In synthesis, side chains of lysineresidues other than the R₅ lysine were protected, as were other reactiveside chains, with selective deprotection following synthesis. Theresulting synthetic growth modulator analog is of the following specificstructure:

and is sometimes called B7A1-K—NS. In the foregoing structure, “Ahx” is6-amino hexanoic acid, sometimes also called “6-Ahx” or “Hex”. Thesingle letters are standard amino acid single letter abbreviations forthe naturally coded amino acids. The chain of SEQ ID NO:9 is grown fromthe alpha and epsilon amine groups of the lysine in the R₅ position.

EXAMPLE 17

B7A1-K—NS was tested in cell osteogenic differentiation studies todetermine the ability of the synthetic growth analog to stimulateosteogenic activity. Referring now to FIG. 11, the induction ofosteogenic differentiation in C2C12 cells with varying concentrations ofB7A1-K—NS in the presence and absence of BMP-2 is illustrated. Treatmentof C2C12 cells with B2A2 alone (up to 10 μg/mL) did not affect theproduction of ALP activity. However, B7A1-K—NS plus BMP-2 results insignificant increases of ALP activity even at normally sub-thresholdconcentrations (100 ng/mL) of BMP-2. C2C12 cells were seeded onto96-well plates, treated with varying concentrations of B2A2 in thepresence (solid bars) and absence (open bars) of BMP-2 at 100 ng/mL. Thecells were incubated for 4 days, and then assayed for ALP activity. ALPactivity was assayed by conversion of para-nitrophenol phosphate (PNPP).B7A1-K—NS had no effect on the induction of ALP activity atconcentrations up to about 10 μg/mL. B7A1-K—NS substantially augmentsALP activity induced by suboptimal amounts of BMP-2 (100 ng/mL). Similarresults were obtaining with C3H10T½ cells.

EXAMPLE 18

A compound of the present invention is synthesized by solid phasepeptide chemistry with the general structure of formula I wherein X is aBMP-2 receptor binding amino acid sequence having the sequenceISMLYLDENEKVVLKNY (SEQ ID NO:8) wherein SEQ ID NO:8 is stepwisesynthesized in parallel from R₂ trifunctional amino acids of formula Iand wherein each R₂ is lysine. The resulting synthetic growth modulatoranalog is of the following specific structure:

In the foregoing structure, “Ahx” is 6-amino hexanoic acid, sometimesalso called “6-Ahx” or “Hex”. The single letters are standard amino acidsingle letter abbreviations for the naturally coded amino acids. The twochains of SEQ ID NO:8 link to lysines in the R₂ position via a peptidebond with the secondary amine of the lysine side chains.

EXAMPLE 19

A compound of the present invention is synthesized by solid phasepeptide chemistry with the general structure of formula I wherein X is aBMP receptor binding amino acid sequence having the sequenceLYVDFSDVGWNDW (SEQ ID NO:10) wherein SEQ ID NO:10 is stepwisesynthesized in parallel from the R₂ trifunctional amino acids of formulaI and wherein each R₂ is lysine. The resulting synthetic growthmodulator analog is of the following specific structure:

In the foregoing structure, “Ahx” is 6-amino hexanoic acid, sometimesalso called “6-Ahx” or “Hex”. The single letters are standard amino acidsingle letter abbreviations for the naturally coded amino acids. The twochains of SEQ ID NO:10 link to lysines in the R₂ position via a peptidebond with the secondary amines of the lysine side chains.

EXAMPLE 20

A synthetic growth modulator analog of the BMP family is synthesized bysolid phase peptide chemistry with the general structure of formula Iwherein X is a BMP receptor binding amino acid sequence having thesequence CAISMLYLDENEKVVL (SEQ ID NO:12) wherein SEQ ID NO:12 isstepwise synthesized in parallel from R₂ trifunctional amino acids offormula I and where R₂ are each lysine. The resulting synthetic growthmodulator analog is of the following specific structure:

In the foregoing structure, “Ahx” is 6-amino hexanoic acid, sometimesalso called “6-Ahx” or “Hex”. The single letters are standard amino acidsingle letter abbreviations for the naturally coded amino acids. The twochains of SEQ ID NO:12 link to lysines in the R₂ position via a peptidebond with the secondary amines of the lysine side chains.

EXAMPLE 21

A compound of the present invention is synthesized by solid phasepeptide chemistry with the general structure of formula I wherein X is aBMP receptor binding amino acid sequence having the sequenceAFYCHGECPFPLADHL (SEQ ID NO:13) wherein SEQ ID NO:13 is stepwisesynthesized in parallel from R₂ trifunctional amino acids of formula Iand wherein each R₂ is lysine. The resulting synthetic growth modulatoranalog is of the following specific structure:

In the foregoing structure, “Ahx” is 6-amino hexanoic acid, sometimesalso called “6-Ahx” or “Hex”. The single letters are standard amino acidsingle letter abbreviations for the naturally coded amino acids. The twochains of SEQ ID NO:13 link to lysines in the R₂ position via a peptidebond with the epsilon amines of the lysine side chains.

EXAMPLE 22

A compound of the present invention is synthesized by solid phasepeptide chemistry with the general structure of formula I wherein X is aBMP receptor binding amino acid sequence having the sequencePFPLADHLNSTNHAIVQTLVNSV (SEQ ID NO:14) wherein SEQ ID NO:14 is stepwisesynthesized in parallel from R₂ trifunctional amino acids of formula Iand wherein each R₂ is a lysine. The resulting synthetic growthmodulator analog is of the following specific structure:

In the foregoing structure, “Ahx” is 6-amino hexanoic acid, sometimesalso called “6-Ahx” or “Hex”. The single letters are standard amino acidsingle letter abbreviations for the naturally coded amino acids. The twochains of SEQ ID NO:14 link to lysines in the R₂ position via a peptidebond with the secondary amines of the lysine side chains.

EXAMPLE 23

A compound of the present invention was synthesized by solid phasepeptide chemistry with the general structure of formula I wherein X is aBMP-2 receptor binding amino acid sequence having the sequenceAISMLYLDENEKVVL (SEQ ID NO:7) wherein SEQ ID NO:7 was stepwisesynthesized in parallel from R₂ trifunctional amino acids of formula Iwhen R₃ is 0 backbone atoms and each R₂ is lysine. The resultingsynthetic growth modulator analog is of the following specificstructure:

and is sometimes called B2A2-K2-NS. In the foregoing structure, “Ahx” is6-amino hexanoic acid, sometimes also called “6-Ahx” or “Hex”. Thesingle letters are standard amino acid single letter abbreviations forthe naturally coded amino acids.

EXAMPLE 24

The compound of Example 2 was tested for specific binding to BoneMorphogenic Protein-2 receptors. Referring now to FIG. 12, results ofsolid phase receptor binding assays utilizing purified receptor/Fcchimeric molecules are illustrated. The chimeras are recombinantconstructs of the soluble ectodomain of various receptor molecules (BMPRand ActivinR isoforms) fused to the carboxyl-terminal of the human IgG1Fc region via a polypeptide liner. ELISA plates were coated with B2A2 orcontrol compounds, soluble chimeric receptor/Fc antibody and quantifiedwith a colorimeteric ELISA. B2A2 was shown to bind preferentially toBMPR-Ib and ActivinR-II, as well as other isoforms in the followingorder: BMPR-Ib=ActR-II>>BMPR-Ia=ActRIIb>BMPR-II. Insulin, used as acontrol, did not bind either B2A2 or BMP-2 (data not shown). Referringnow to FIG. 13, B2A2 binding to purified BMP-2 receptor/Fc chimericmolecules in varying concentrations of BMP-2 is illustrated. BMP-2 addedin large molar excess with the receptors blocked binding to B2A2. WhenBMP-2 was added in varying concentrations, the resulting displacementcurve suggests two-stage binding kinetics of B2A2 to BMPR-Ib.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverin the appended claims all such modifications and equivalents. Theentire disclosures of all references, applications, patents, andpublications cited above are hereby incorporated by reference.

What is claimed is:
 1. A compound of Formula II:

wherein: X is a peptide sequence selected from the group consisting ofSEQ ID NOs: 7-14; R₁ is hydrogen, such that the terminal group is —NH₂;R₆ is an amino acid; R₅ is is a trifunctional amino acid; R₄ is NH₂ atthe carboxyl terminal group; Y is Ahx-Ahx-Ahx, wherein Ahx is6-aminohexanoic acid; and Z is a peptide sequence selected from groupconsisting of SEQ ID NO 3-6.